Leishmania sp. protozoa are exposed to diverse environments as they cycle between an insect vector and a mammalian host. Within these diverse environments the parasites exist in different developmental forms, uniquely adapted for survival in their specific host. Part of their survival mechanisms involves the developmental expression of surface antigens. We have found that the amount of the major surface protease (gp63) in Leishmania donovani chagasi (Ldc) promastigotes increases 10-fold as they develop from a less infectious form during early logarithmic growth to a more virulent form during stationary phase of in vitro cultivation. This process probably mimics the natural development of promastigotes to an infectious state in the gut of their sandfly vector. The increase in gp63 is accompanied by differential expression of three RNA transcripts from different gp63 gene classes, distinguishable by unique 3' untranslated regions (UTRs) (Manuscripts 1 and 2, Appendix). The goal of the proposed work is to delineate the molecular mechanisms directing the expression of the different gp63 genes during the in vitro developments of Ldc promastigotes. Gp63 is encoded by a family of tandemly repeated genes on a single chromosome. We will begin our studies by clarifying the genomic organization of the three gp63 gene classes in Ldc, and identifying flanking and intercistronic sequences that are potential regulatory elements. We will examine the transcription rates of the three gp63 gene classes and the potential contribution of the unique 3'UTRs to the steady state levels of the gp63 RNAs at different growth stages of promastigote cultures. We will explore the roles of DNA- and RNA-binding proteins in regulating the rates of gp63 gene transcription and gp63 RNA degradation. The functional roles of the protein products of individual gp63 genes will be addressed by replacement, insertion and disruption of these genes in cloned promastigotes. Gp63 mutants will be assayed for intracellular survival, virulence and attachment to macrophage receptors. Finally, we will determine if protein is expressed from each of the unique gp63 RNAs. We expect that the collective molecular mechanisms that we find to regulate the expression of the gp63 gene family will be applicable to a general understanding of developmental regulation in Trypanosomatid protozoa.